First Report of a Novel Strain of Tomato yellow leaf curl virus Causing Yellow Leaf Curl Disease on Cluster Bean in Pakistan

In 2015-16, during field surveys of Sindh and Punjab provinces of Pakistan, characteristic Begomovirus-like symptoms of leaf yellowing and curling were observed on Cyamopsis tetragonoloba. C. tetragonoloba, also known as cluster bean or guar, is a leguminous crop commonly cultivated in India and Pakistan. To test for possible begomoviruses, one symptomatic plant and one asymptomatic plant was collected from Sindh and two symptomatic and one asymptomatic plant was collected from Punjab. Total DNA was extracted from leaf tissue of all plants using a CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1987). Southern blot hybridization was performed using a DIG labeled probe to the Tomato yellow leaf curl virus (TYLCV) C1 gene. A strong hybridization signal was observed in symptomatic plants only. To confirm the association of TYLCV, DNA from one positive sample was used in rolling circle amplification (RCA) for viral DNA enrichment using phi29 DNA polymerase (Thermo Fisher Scientific, Waltham, MA, U.S.A.). The RCA product was purified and sequenced by the Illumina MiSeq system. The Illumina NeoPrep automation system was used with the Illumina TruSeq Nano DNA Library Prep Kit (NP-101-1001). Adapters were trimmed using MiSeq Reporter Software and sequencing data were analyzed using CLC Genomics Workbench 7.5 (https://www.qiagenbioinformatics.com). The total number of raw reads obtained was 264,578. De novo assemblies were prepared following the standard quality parameters (quality score: 0.001 and Phred score: 30). Sequence analysis resulted in one contig corresponding to the full-length genome of TYLCV (∼2800 nucleotides) with a coverage of 107,574 reads. An NCBI BLASTn of the contig confirmed the presence of TYLCV (deposited in GenBank as KX710157). A BLASTn analysis of the contig sequence showed that TYLCV from Pakistan was 92% identical to TYLCV reported in 2005 on tomato from Al-Batinah, Oman. TYLCV PCR primers were designed from the contig sequence as follows: TYLCV-PK-F 5′-CGACCAGTCTGAGGCTGTAAT-3′ and TYLCV-PK-R 5′-CCCTTTAATTTGAATGGG-3′. DNA from each symptomatic sample tested positive for TYLCV by PCR and the amplification products (511 bp) were cloned into a T/A cloning vector (pTZ57R/T; ThermoFisher Scientific). Clones were Sanger sequenced. Sequences were assembled using Lasergene software (DNA Star Inc.) and a consensus sequence was deposited in GenBank as KY769274. A BLASTn analysis of the consensus sequence showed 97% identify to TYLCV-Iran isolate from tomato in Iran. The species and strain demarcation of TYLCV was confirmed using the Sequence Demarcation Tool (SDT) program (http://web.cbio.uct.ac.za/∼brejnev/), following the taxonomic criteria of begomoviruses (Brown et al. 2015). A MUSCLE alignment was performed using default settings of SDT, using all 347 available sequences of TYLCV from the ICTV (http://www.ictvonline.org/virustaxonomy.asp). SDT-MUSCLE analysis indicated that the TYLCV, found on cluster bean, is a novel strain, to which we purpose the name TYLCV-Pakistan (TYLCV-PK), following the standard nomenclature for begomoviruses (Brown et al. 2015). TYLCV has been detected in several countries and is a serious threat to economically important crops such as tomato and cucurbits (Mabvakure et al. 2016). The occurrence of TYLCV-PK on cluster bean indicates the increasing geographical and host range of TYLCV. This is the first report of TYLCV-PK, a novel strain of TYLCV; this is also the first report of TYLCV infecting cluster bean or any other crop in Pakistan. © 2017, American Phytopathological Society. All rights reserved.